Fine structure of early human embryos frozen with 1,2 propanediol

Previous studies by a French group (Fertil Steril 44:645–651, 1985) have shown that two-to eight-cell human embryos can survive slow freeze-thawing with propanediol in a biological freezer. These embryos were assessed for morphological appearance by phase-contrast microscopy. We assessed the structure of 25 frozen-thawed one- to 12-cell embryos, obtained from our in vitro fertilization (IVF) and GIFT programmes, by phase-contrast and electron microscopy, using the same method of cryopreservation. One-fourth of the embryos examined had all cells intact, and more than one-half the embryos had over 50% of their cells well preserved. Some of these embryos had unequal blastomeres and cytoplasmic fragments. Ultrastructural assessment revealed good preservation of fine structure in the intact blastomeres of all embryos and maintenance of cell-to-cell contacts. Most cytoplasmic organelles, cell membranes, and nuclei were well preserved compared to nonfrozen controls. The cells that were cryoinjured showed varying degrees of disorganization of the cell membrane, cytosol, and cellular membranes, including swelling and disruption of the nuclear envelope. Disruption of the zona was somewhat rare. Small cytoplasmic fragments were less prone to cryoinjury than blastomeres. The use of propanediol for embryo cryopreservation seems to be feasible; frozen embryos with more than 50% cells intact have produced 10 pregnancies after embryo transfer (Fertil Steril 46:268–272, 1986). Replacement of 17 frozen embryos in seven patients has resulted in a twin pregnancy in Singapore. However, the effects of freezing on the mitotic spindles of embryonic cells need to be investigated further.     

Sperm-oocyte membrane fusion in the human during monospermic fertilization

Sperm-oocyte membrane fusion has been observed during monospermic fertilization of a human oocyte in vitro. Women were stimulated with both clomiphene citrate and human menopausal gonadotropin and were given human chorionic gonadotropin before a LH-surge. Twelve oocytes, collected at laparoscopy from six women who became pregnant by IVF, were allowed to mature for 7–14 hours in vitro and inseminated with preincubated sperm, fixed between 1–3 hours after insemination, and examined by transmission electron microscopy. Membrane fusion had occurred in one ovum 2 hours after insemination, and the oocyte had resumed maturation and was at anaphase II of meiosis. Cortical granules had been exocytosed, and some of their contents were visible at the surface close to the oolemma all around the oocyte. The sperm that fused with this oocyte was acrosome-reacted and had been partly incorporated into the ooplasm, while the anterior two-thirds of its head was phagocytosed by a tongue of cortical ooplasm. Membrane fusion had occurred between the oolemma and the plasma membrane overlying the postacrosomal segment of the sperm head, posterior to the equatorial vestige. Sperm chromatin had not decondensed, and serial sections revealed a midpiece attached to the basal plate and a tail located deeper in the ooplasm, all devoid of plasma membrane. Supplementary sperm penetrating the inner zona, approaching the perivitelline space, had undergone the acrosome reaction but had a persistent vestige of the equatorial segment of the acrosome with intact plasma membrane. Evidence of sperm chromatin decondensation was seen in other oocytes, 3 hours after insemination, which were at telophase II of meiosis. Eight oocytes penetrated by sperm were monospermic, while four were unfertilized. The general pattern of sperm fusion and incorporation appears to conform to that seen in most other mammals. The study also reveals that sperm have to complete the acrosome reaction before fusing with the egg.     

The human pronuclear ovum: Fine strcture of monospermic and polyspermic fertilization in vitro

The fine structure of pronuclear ova (monospermy and polyspermy) and one-cell embryos has been investigated in our IVF programme. Sixteen oocytes were collected at laparoscopy after appropriate hormonal stimulation and were matured and fertilized in vitro by methods that have given rise to normal pregnancies. Pronuclear ova showing monospermic fertilization had two vesicular pronuclei surrounded by aggregations of cellular organelles. The male pronucleus was closely associated with a sperm axoneme, while the female pronucleus was dismantling its envelope and condensing its chromatin ahead of its counterpart in late pronuclear ova. Each pronucleus had dispersed chromatin, dense compact nucleoli, and intranuclear annulate lamellae. Smooth endoplasmic reticulum, annulate lamellae, Golgi complexes, and mitochondria formed a conspicuous part of the perinuclear ooplasm. The one-cell embryos were either in syngamy or in the process of undergoing first cleavage. Positive evidence of cortical granule release and second polar bodies were detected in the perivitelline space. A block to polyspermy seemed to operate at the level of the inner zona. Dispermic and polyspermic ova had 3–16 pronuclei resembling those of monospermic ova and had sperm tails in the ooplasm. Sperm were also seen penetrating the inner zona and were occasionally found in the perivitelline space. Incomplete cortical granule release and early signs of cytoplasmic fragmentation were noted in polyspermic ova. Both normal and abnormal features of these ova are reported and compared with pronuclear structure in vivo and in vitro.     

Ultrastructural observations on cortical granules in human follicular oocytes cultured in vitro

The fine structure, distribution, and fate of cortical granules in human oocytes cultured in vitro are reported. Follicular maturation in women with blocked Fallopian tubes was induced by clomiphene citrate and human chorionic gonadotropin, and preovulatory eggs were obtained by improved methods of laproscopy and oocyte recovery. These oocytes were then inseminated and cultured in a modified Ham's F10 medium for 3 to 72 hr to assess their fertilizability. Cortical granules were observed in all 17 unfertilized oocytes investigated, which had completed various stages of meiotic maturation. A marked increase in their numbers was observed in oocytes cultured for 3 to 6 hr. There was no evidence of spontaneous cortical granule release in any of the oocytes studied. It is concluded that cortical maturation expressed by proliferation of cortical granules is as significant a criterion as nuclear maturation in assessing maturity and fertilizability of oocytes cultured in vitro. A short sojourn in culture before insemination could improve chances of normal fertilization and embryo development, which has been recently achieved in our laboratory.     

The effects of ultrarapid freezing on meiotic and mitotic spindles of mouse oocytes and embryos

Preovulatory mouse oocytes and 2-cell embryos were frozen with dimethyl sulfoxide and propanediol by an ultrarapid method. The survival of frozen oocytes was low (33–34%) compared to that of 2-cell embryos (78–79%) with either cryoprotectant. Development to blastocysts after postthaw culture was about 7–15% for oocytes and 79–80% for the embryos. Ultrarapid freezing preserves cell structure quite well as revealed by electron microscopy, but meiotic oocytes and late 2-cell embryos undergoing mitosis showed evidence of spindle disorganization involving loss or clumping of microtubules resulting in some scattering of chromosomes. Embryos developed from frozen eggs showed clear evidence of micronuclear formation and incomplete incorporation of chromosomal material into main nuclei. These experiments confirm our observations on freezing of human oocytes and show that spindle microtubules are sensitive to freeze-thawing and that cryopreservation could cause chromosomal aberrations during early development. A cautious approach to the introduction of oocyte freezing in human in vitro fertilization (IVF) programs is advocated.     

Degeneration of monoamine nerves in anterior byssus retractor muscle of Mytilus induced by 5,6-dihydroxytryptamine

Summary Preliminary ultrastructural studies on the effects of 5,6-Dihydroxytryptamine (5,6-DHT) on the anterior byssus retractor muscle (ABRM) of Mytilus show degeneration of 2 types of monoaminergic nerves after 10 days of drug treatment. One type contained large granular vesicles (560–1,680 Å) while the other had small granular vesicles (200–640 Å). These axons may possibly represent serotonergic and dopaminergic nerves, thought to innervate this muscle.Two other types of profiles seemed to be unaffected by the drug. One conforms to cholinergic nerves while the other has a predominance of large opaque vesicles (1,200–2,500 Å). The significance of these findings is discussed in the light of recent observations on the neurotoxic effects of 5,6-DHT on vertebrate and molluscan nerves.The author is grateful to Professor G. Burnstock for research facilities and Professor B. M. Twarog for advice and encouragement. This work was supported by the Ramaciotti Foundation     

Centriolar dynamics in the bovine embryo: inheritance and perpetuation of the sperm centrosome during fertilization and development

Summary Inheritance of the centrosome (centriole) and its behaviour during fertilization and embryogenesis of cattle is presented. The bovine embryo follows the human pattern of centriole behaviour, which is common to most animals including large mammals. Thus, most mammals obey Boveri's rule of paternal centrosomal inheritance and perpetuation, whereas the mouse is an exception to the rule, showing maternal inheritance. The sperm centrosome was traced from fertilization to the hatching blastocyst stage in the cow and its presence was confirmed at every stage of cleavage, as reported in the human. It is concluded that the bovine embryo is a more appropriate model than the mouse for research in fertilization and assisted-reproduction technology.     

Subzonal transfer of multiple sperm (MIST) into early human embryos

Microinsemination sperm transfer (MIST) is a technique whereby sperm are transferred into the perivitelline space (PVS) with the aid of a micromanipulator. MIST is now used to investigate whether blastomere membranes of early human embryos are capable of fusing with the sperm as in the metaphase II oocyte. Between 10 and 30 sperm were transferred into 11 donated human embryos between pronuclear and 16 cell stage. After culture for 6-24 hr in vitro, the embryos were fixed for transmission electron microscopy (TEM). Both acrosome-intact and acrosome-reacted sperm were located in the PVS and between blastomeres. Sperm in the PVS were sometimes penetrating the inner regions of the zona. Sperm-blastomere membrane fusion was not observed, but sperm tail incorporation by phagocytosis was occasionally evident. Sperm heads incorporated into blastomeres were often located in membrane-bound vesicles. Both acrosome-intact and acrosome-reacted sperm heads were found in vacuoles. Acrosome-reacted sperm heads were lying passively in vacuoles or were undergoing degenerative changes at their surfaces. Sperm chromatin decondensation was not observed in any of the sperm heads that were detected in the blastomeres. The evidence presented clearly shows that sperm heads are incapable of expanding their chromatin to form typical male pronuclei following MIST into early human embryos.     

Approximate Michaelis-Menten kinetics displayed in a stochastic model of cell-mediated cytotoxicity

A nonlinear continuous-time Markov chain describing a two-step process of cytolytic cells binding to target and the subsequent lysis and release of label is shown to have kinetics which resemble standard enzyme-substrate kinetics. The Michaelis-Menten saturation function is found as a special case resulting when the target population is in excess. A comparison theorem for the pseudo-steady-state distribution Π is constructed to enable examination of that distribution whose expected value E and variance V satisfy - K_mE + (C_T − E)(T_T − E) + V = 0, where K_m is the Michaelis half-saturation constant and C_T and T_T are the initial populations of the two cell types. Using Π as an initial condition, the release of label process is examined. The main result is that the fraction of specific release, ƒ, has the approximate form when T_t is large, so that a nonlinear regression procedure is appropriate for the determination of the parameters.